Immunomodulating Nanoparticulate Composition

ABSTRACT

The present invention relates to a preferably nebulizable pharmaceutical composition comprising a pharmaceutically acceptable protein-based nanocarrier preferably in the size range 150 to 300 nm and a preventative or therapeutic amount of an active agent for use in the prevention and/or treatment of an allergic and/or inflammatory disease of the lower airways in a mammal. Preferably, the active agent is a CpG oligodeoxynucleotide (CpG-ODN), and preferably the composition exhibits a prolonged clinical effect.

This application takes benefit of the foreign priority of application EP11001858 filed on Mar. 7, 2011.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition comprisinga pharmaceutically acceptable polymerized protein-based nanocarrierpreferably in the size range 150 to 300 nm and a preventative ortherapeutic amount of an active agent for use in the prevention and/ortreatment of an allergic and/or inflammatory disease of the lowerairways in a mammal. Preferably, the active agent is an oligonucleotideand/or an oligodeoxynucleotide (ODN) which is effective for use in theprevention and/or treatment of an allergic and/or inflammatory diseaseof the lower airways, which is preferably selected from the groupconsisting of guanidine phosphodiester cytosine (CpG) ODN class A, classB and/or class C. Said pharmaceutical composition preferably has aprolonged effect in the prevention and/or treatment of an allergicand/or inflammatory disease of the lower airways, preferably at asurprisingly low dose.

The allergic or inflammatory disease is preferably associated withelevated serum and/or pulmonary interleukin (IL-10) levels, and tomethods for the production of said composition for use in the preventionand/or treatment of allergic and/or inflammatory diseases of the lowerairways.

BACKGROUND OF THE INVENTION

The overwhelming necessity for effective and causal treatment ofallergic diseases is well known in the art. Within the large group ofallergic diseases, which are in general characterized by versatilehypersensitivity type I reactions caused by a disordered activation ofthe immune system and which comprise allergic rhinitis, asthmabronchiale, atopic eczema, anaphylaxis, insect venom, drug allergies,food allergies and multiple allergies, allergic airway diseases such asasthma are among the most prevalent ones to impair quality of life andlife expectation. In the last decades, allergic airway diseases havedramatically increased in the northern hemisphere of industrializedcountries humans and domestic animals such as horses (Kline 2007;Braun-Fähränder 2009). As of 2009, 300 million people worldwide wereaffected by asthma leading to approximately 250,000 deaths per year. Itis estimated that asthma has a 7-10% prevalence worldwide with a greatdisparity in prevalence worldwide across the world (as high as a 20 to60-fold difference). A trend toward more developed and westernizedcountries having higher rates of asthma was observed.

Depending on the severity, allergic airway diseases such as asthma areassociated with inflammation and airway obstruction. However, even ifsome symptoms might be comparable, the underlying principles causing thedisease are different from chronic obstructive pulmonary disease (COPD).Nevertheless, a medication to both control allergy and to reduceinflammation would be of great general advantage.

In present medication of asthma one can be distinguished between fastacting and long term control. Short acting beta 2-adrenoceptor agonists(SABA), such as salbutamol are the gold standard in treatment of asthmasymptoms. Anticholinergic medications such as ipratropium bromideprovide addition benefit when used in combination with SABA in thosewith moderate or severe symptoms. Anticholinergic bronchodilators are analternative if a person cannot tolerate a SABA.

For long term control, glucocorticoids are considered the most effectivetreatment available. Inhaled forms are usually used except in the caseof severe persistent disease, in which oral steroids appear appropriate.Inhaled formulations may be used once or twice daily, depending on theseverity of symptoms. Long acting beta-adrenoceptor agonists (LABA) haveat least a 12-hour effect. They are however not to be used without asteroid due to an increased risk of severe symptoms and are thuschallenged for additive value. Leukotriene antagonists such asmontelukast or zafirlukast are an alternative to inhaledglucocorticoids, but are second line at present. Mast cell stabilizerssuch as cromolyn sodium are another but less potent alternative toglucocorticoids. Anti-immunoglobulin E (IgE) monoclonal antibodiesconstitute a relatively new but not yet broadly established potentiallycausal from of antiallergic medication.

Despite such advanced knowledge and various available mediationregimens, the increasing frequency of asthma is alarming. Rates ofasthma have increased significantly between the 1960s and 2008 with 9%of US children suffering from asthma in 2001, compared with just 3.6% in1980. The World Health Organization (WHO) reports that today 10% of theSwiss population suffers from asthma today compared with just 2% some25-30 years ago. Thus, the establishment of alternative causalmedication strategies is highly desirable and required.

Allergic airway diseases are not restricted to humans. Like humanasthma, recurrent airway obstruction (RAO) in horses is considered amultifactor allergic airway hypersensitivity reaction elicited byenvironmental exposure to potential allergens (Robinson 2001) andheritable components (Gerber et al. 2009). Equine RAO has become one ofthe most common airway diseases (Fey 2006). Housing of horses in stableswith permanent exposure to potentially allergenic organic and inorganicparticles was reported to be a major trigger factor (Schmallenbach etal. 1998; Robinson 2001; Millerick-May 2009). Keeping horses on pastureleads to improved clinical signs, however, complete avoidance ofallergens is not always possible (Robinson 2001). The permanentinhalation of various antigens from moldy hay, mite dust as well asendotoxin, β-glucan and other organic and inorganic particles causesairway neutrophilia and inflammation with a mixed Th1/Th2 immuneresponse (Horohov et al. 2005; Cordeau et al. 2004). Although theclinical signs of RAO were well defined (Robinson 2001), immunologicalmechanisms are still controversy discussed. However, a predominant Th2allergic response was recently presumed (Horohov et al. 2009). Inseveral studies, signs of predominant Th2 response such as high IL-4,IL-5 in bronchoalveolar lavage fluid (BALF) of RAO horses exposed toantigens were reported (Lavoie et al. 2001; Cordeau et al. 2004). Due topersistent chronic inflammatory reaction in small airways of theaffected horses, Th1 participation was also confirmed (Ainsworth et al.2003).

Unmethylated Cytosin-Phosphate-Guanin-Oligodeoxynucleotides (CpG-ODN)were described as effective immune stimulating agents to cause a Th2/Th1immune shift (Kline 2007). This shift further promoted animmunoglobuline isotype switch from IgE to IgG2 (Bohle 2002). Theprevalence of IgE is a matter of ongoing debate (Halliwell et al. 1993;Marti 2009). In addition, a shift from pro-allergy mediating IL-4, IL-5and IL-13 Th2 cytokines towards pro-inflammatory IFN-γ and IL-12 Th1cytokines was discussed. Anti-inflammatory and antiallergic propertiesof Th2 cytokine IL-10 turned out to be of interest. Particularly, IL-10producing T regulatory cells (Tregs) and its balance towards Th2 cellsseems to play an important role in immune homeostasis (Akdis et al.2004; Lloyd and Hawrylowicz 2009). Thus, one problem to be solved by thepresent invention is the clarification whether alternatively or inaddition to the present conventional symptomatic therapy the possibilityto modulate cytokine level in order to avoid development of an allergichypersensitivity constitutes a promising option for use in the treatmentof allergic and/or inflammatory lower airway diseases.

However, so far no decisive IL-10 regulating nanoparticulate compositioncomprising CpG-ODN has been proposed for use in therapy in allergic andinflammatory airway diseases, especially not my means of inhalation.Hence, there is a need for immunomodulating nucleic acids in ananoparticulate composition for use in the treatment of allergic and/orinflammatory airway diseases such as asthma and RAO.

Three distinctive CpG classes (A-, B- and C-class) with varyingimmunologic effects were previously investigated (Krieg 2002). The keypattern-recognition receptor for these “danger signals” is the Toll-Likereceptor 9 (TLR-9) which is located in endosomes (Krieg 2002). Recently,equine TLR-9 was detected in monocytes, airway epithelial cells,capillary endothelium in the lungs and pulmonary intravascularmacrophages (Schneeberger et al. 2009). Furthermore, species-specificimmune stimulation depending on CpG motifs has been determined (Hartmannand Krieg 2000; Rankin et al. 2001). US2004/0132682 claims aninteresting method of treating inflammatory lung disease withsuppressors of CpG-ODNs. However, these inflammatory lung diseases wereexplicitly not of allergic origin. The person of ordinary skillappreciates that the immunologic processes underlying allergic andnon-allergic inflammatory inflammation of the lung are distinctive, e.g.concerning the involvement of neutrophils versus eosinophils.

Nevertheless, a promising immunotherapeutic strategy against allergicconditions of the upper airways including the nose as well as of theeyes already entering human clinical phase IIa studies involved CpG-ODN(Senti et al. 2009) as adjuvant, i.e. a synthetic analog of naturalmicrobial CpG-ODN (Krieg 2002). This TLR9 agonist demonstrated someefficiency in treatment of allergic diseases if combined as an adjuvantwith specific allergens due to CpG-DNA's potential to cause a Th2/Th1shift (Vollmer and Krieg 2009, Kline 2007, Krieg 2002) which wasassociated with a downregulation of proallergic Th2 cytokines (IL-4,IL-5, IL-13) and an upregulation of antiallergic Th1 cytokines (IFN γ,IL-12) and an immunoglobulin isotype switch from allergy mediating IgEto IgG (Bohle 2002). U.S. Pat. No. 6,429,199 claims such CpGs asadjuvants in immunotherapy. The role of CpGs as adjuvants in order tostimulate the immune system is further supported by U.S. Pat. No.7,488,490. However, despite for treatment of non-allergic lung diseases(US2004/0132682) such prior art rather teaches away from the employmentof CpG-ODNs in monotherapy, especially for inhalation therapy, asinhalation of CpG-ODNs without immunotherapy did not result in areduction of allergic clinical symptoms in a clinical trial (Fonseca etal 2009). Moreover, prior art is silent on an immunmodulating actionbased on IL-10 stimulation and especially, nanoparticles in no form aredisclosed as eligible for use in related immunomodulating therapy,neither in general nor specifically for inhalative administration.

Pulmonary delivery as a non-invasive route of drug administration stillconstitutes a vivid research field in the treatment and diagnosis ofrespiratory and non-respiratory diseases (Smola et al. 2008). However,nanoparticulate delivery is still a partly unexplored field.

As an example for protein-based nanoparticles, gelatin nanoparticles(GNPs) from gelatin type A obtained from porcine skin were found to beimmunologically inert and the use of GNPs showed amplified CpG-relatedTLR-9 activation for use as adjuvant in anticancer immunotherapy(Zwiorek et al. 2008). However, none such composition was ever evaluatedin reducing the immune system's activity, for example for effective usein the treatment of allergic and/or inflammatory diseases. In detail,one problem to be solved is to identify a composition comprising theoptimal immunomodulating agent to trigger specific immunomodulatingeffect such as Th2/Th1 shift and IL-10 stimulation and to reduce, forexample, inflammation in clinical allergic symptoms known to the skilledperson

Compressor or jet nebulizers apparently are not suitable for nebulizingcompositions comprising fragile nucleic acid such as CpG-ODNs even ifjet nebulized protein-based nanoparticles such as from gelatin gavepromising results such as enabling targeted lung cancer delivery ofchemotherapeutic agent cisplatin (Tseng et al. 2007; Tseng et al. 2009).However, these compositions were much more stable than any nucleic acidcomprising nanoparticulate composition. Tseng determined the mediandroplet size and lung deposition was proved in vivo, but no furthercharacterization of the data of the nebulization process itself wasgiven. US2005/019270 describes a method to nebulize spray-dried GNPsleaving the skilled person in doubt whether such methods destroyssensitive payload such as CpG-ODN. Against this technical background andthe fact that the frequency of allergic and inflammatory airway diseasesis still increasing, the problem is that there remains a tremendous needfor new, long-lasting and efficient composition for use in causalprevention and/or treatment of allergic and inflammatory airwaydiseases.

SUMMARY OF THE INVENTION

In the present invention, the inventor surprisingly found thatnanoparticulate compositions comprising various CpG ODNs andprotein-based nanoparticles, respectively, are eligible for use in theprevention and/or treatment of allergic and/or inflammatory diseases ofthe lower airways. Moreover, the inventors found that such a compositionexhibits a long-lasting therapeutic effect even after treatment hasceased. In addition, one single CpG ODN proofed to be sufficient as soleand main active ingredient in such a composition, preferably in asurprisingly low dose.

This was not taught or suggested by the prior art which, as explainedabove, revealed that certain ODNs, in particular CpGs, have distinctiveeffects on immune cells and are potentially useful as adjuvants for usein treatment of cancer and hypersensitivities even though it was knownthat CpGs can be used in compositions together with nanoparticles.

In a first aspect, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable polymerizedprotein-based nanocarrier in the size range preferably between 150 to300 nm and a preventative or therapeutic amount of an active agent,

for use in the prevention and/or treatment of an allergic and/orinflammatory disease of the lower airways in a mammal,

wherein the active agent is an oligonucleotide and/or an ODN which iseffective for use in the prevention and/or treatment of an allergicand/or inflammatory disease of the lower airways,

wherein the oligodeoxynucleotide is selected from the group consistingof guanidine phosphodiester cytosine (CpG) ODN class A, class B and/orclass C,

and wherein the active agent is coupled to the polymerized protein-basednanoparticle in a manner wherein the active agent maintains itspreventive and/or therapeutic activity.

In a preferred embodiment of said aspect, the polymerized protein-basednanocarrier the pharmaceutical is a gelatin nanoparticle, an albuminnanoparticle, a legumine nanoparticle, a gliadine nanoparticle, anelastinlike polypeptide nanoparticle, a beta-galactoglobulinenanoparticle and/or a silk protein nanoparticle

In a further preferred embodiment of said aspect, the pharmaceuticalcomposition is a nebulizable aqueous dispersion or a nebulizable aqueousdispersion made from a lyophilisate.

In another preferred embodiment of said aspect, the aqueous dispersionis nebulized by a vibrating mesh nebulization device,

wherein the resulting droplet size is preferably in the size rangebetween 1 to 5 μm,

wherein the respirable droplet fraction is 50 to 100%,

wherein the nebulization efficiency is more than 95%,

wherein the nanoparticle concentration within the aqueous dispersion ispreferably in the range between 1.0 to 2.0 mg/ml,

wherein the dispersant in the aqueous dispersion is highly purifiedwater with a conductivity below 0.55 μS/cm or a physiological 0.9% NaClsolution,

wherein the aqueous dispersion has a viscosity in the range between 0.85and 1.1 mPa/s.

and wherein the nebulizable aqueous dispersion is nebulized by avibrating mesh nebulization device at an output rate over 0.5 ml/min.

In yet another preferred embodiment of said aspect, said the comprisedoligonucleotide and/or an ODN is administered at a dose of 0.0001 to 2mg per kg body weight, preferably in a range between 0.0001 and 0.01mg/kg body weight, most preferably in a range between 0.0002 and 0.001mg/kg body weight.

Preferably the CpG ODN comprises a central palindrome CGmotif-containing sequence flanked by a polyG sequence and a chimericalphosphodiester/phosphorothioate backbone.

In a preferred embodiment of said aspect, said CpG ODN is selected fromthe group consisting of 5′-CTG GTC TTT CTG GTT TTT TTC TGG-3′ (SEQ IDNO: 1), 5′-G*G*G GGA CGA TCG TCG*G*G*G*G*G-3′ (SEQ ID NO:2) wherein *indicate phosphorothioesters although a full phosphorothioate backboneis also disclosed for SEQ ID NO:2 in the present invention, 5′-TCG CGTGCG TTT TGT CGT TTT GAC GTT-3′ (SEQ ID NO:3), 5′-TCG TCG TTT TGT CGT TTTGTC GTT-3′ (SEQ ID NO:4), 5′-TCG TCG TTT TCG GCG CGC GCC G-3′ (SEQ IDNO:5), 5′-TCG TCG TCG TTC GAA CGA CGT TGA T-3′ (SEQ ID NO:6),5′-GGTGCATCGATGCAGGGGGG-3′(SEQ ID NO:7), 5′-GGGGGGGACGATCGTCGGGGGG-3′(SEQ ID NO:8), 5′-GGGGGGGGACGATCGTCGGGGGGG-3′ (SEQ ID NO:9),5′-GGGGGGGGGACGATCGTCGGGGGGGG-3′ (SEQ ID NO:10),5′-GGGGGGGGGGACGATCGTCGGGGGGG-3′ (SEQ ID NO:11),5′-GGGGGGGGGGGACGATCGTCGGGGGGGGGG-3′ (SEQ ID NO:12),5′-GGGGGGGGGGGGACGATCGTCGGGGGGGGGGG-3′ (SEQ ID NO:13), and/or5′-GGGGGGGGGGGGGACGATCGTCGGGGGGGGGGGG-3′ (SEQ ID NO:14) and/or aderivative thereof comprising a chimericalphosphodiester/phosphorothioate backbone or a full phosphorothioatebackbone. SEQ ID NO 2, 7, 8, 9, 10, 11, 12, 13 and 14 belongstructurally to a common group of A-class CpG ODNs with altered G tailsand backbones and are especially preferred.

In another preferred embodiment of said aspect, said CpG induces therelease of antiallergic cytokine interleukin-10 and reduces theprevalence of proallergic and proinflammatory cytokines such as IL-4

In yet another preferred embodiment of said aspect, the mammal is ahuman, a primate, or a domestic animal or an animal for production,preferably a horse.

In still another preferred embodiment of said aspect, wherein the amountof oligonucleotide and/or an ODN is 0.1 to 10 wt %, preferably 1 to 7.5wt % and more preferably 5 wt % in relation to the nanoparticle mass.

In a further preferred embodiment of said aspect, the pharmaceuticalcomposition further comprises excipients selected from the groupconsisting of buffer salts, surfactants, lyoprotectors, dyes, chelatingagents and/or radionuclides.

In another preferred embodiment of said aspect, the pharmaceuticalcomposition is administrated to an individual via inhalation, wherein avibrating mesh nebulization device is combined in such a way with aninhalation spacer in such a manner that the individual can inhale thenebulized pharmaceutical composition from the inhalation spacer, andwherein the vibrating mesh nebulization device is attached to theinhalation spacer, so that the nebulized pharmaceutical composition isquantitatively available at the inhalation spacer's top part nasaloutlet.

In a further preferred embodiment, the composition of the presentinvention induces a prolonged clinical effect by reducing or inhibitingallergic and/or inflammatory symptoms and/or events in the lower airwaysand/or in the general state of health of the treated individual.

In yet still another embodiment of said aspect, the pharmaceuticalcomposition is administered 1 time per day, 2 times per day, 3 times perday, 4 times per day, 5 times per day, 6 times per day, 1 time per week,2 times per week, 3 times per week, 4 times per week, 5 times per weekor 6 times per week, 3 times per months, 2 times per months or 1 timeper month.

Preferably, the pharmaceutical composition of the present invention isadministered in a cycle comprising a period of 3 to 14 d with 3 to 7applications of the composition of claim 1, followed by an applicationfree period of 7 to 84 d. The cycle is optionally succeeded by 1 to 100further cycles, preferably by 1 to 5 cycles. The person skilled in theart is able to adopt the type or amount of cycles to the patient'sindividual needs.

In another preferred embodiment of said aspect, the pharmaceuticalcomposition comprises a further antiallergic and/or anti-inflammatoryactive agent selected from the group consisting of glucocorticoids,H1-antagonists, leukotriene antagonists, mast cell stabilizers beta2adrenergic receptor agonists and/or anticholinergic agents.

In a second aspect, the present invention provides a method for theproduction of the pharmaceutical composition comprises the steps ofcontacting the nanocarrier with the active ingredient in a ratio ofpreferably 90 to 99% nanocarrier to 1 to 9% active agent in apharmaceutically acceptable medium, sensitive mixing, incubating forpreferably 1.5 to 6 h optionally at a shaking rate of preferably 200 to800 rounds per minute and 20 to 25° C., optionally purification bycentrifugation at 10000 to 18000 g and adjusting the concentration to anebulizable nanosuspension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Mass deposition of fluorescent labeled GNPs in the twin-stageimpinger in dependence of the applied aerosolization device. Active VMdevices surprisingly turn out to be preferred

FIG. 2: IL-10 expression from BALF cells from healthy and RAO-affectedhorses with different CpG classes. BAL cells derived from RAO-affectedhorses (individuals n=7, measuring points n=3) (black bars) or healthhorses (individuals n=5, measuring points n=3) (white bars) wereincubated with 13.5 μg of soluble CpG-ODN (A) or GNP-bound CpG-ODN(CpG-GNP) (B). Cells from healthy horses secreted significantly higheramounts of IL-10 than those of RAO-affected horses (P=0.0047). CpG 2216A triggered the highest IL-10 release for both cells groups. Depictedvalues are group means±S.D. and corrected by subtraction of individualnegative controls (untreated BAL cells).

FIG. 3: IL-4 expression from BALF cells from healthy and RAO-affectedhorses with different CpG classes. BAL cells derived from RAO-affectedhorses (individuals n=7, measuring points n=3) (black bars) or healthhorses (individuals n=5, measuring points n=3) (white bars) wereincubated with 13.5 μg of soluble CpG-ODN (A) or GNP-bound CpG-ODN(CpG-GNP) (B). Cells from healthy horses secreted significantly loweramounts of IL-4 than those of RAO-affected horses (P=0.001) when treatedwith GNP-bound CpG-ODN (CpG-GNP) (B). Incubation with soluble CpG-ODNdid not result in such significantly distinguishable release (A).Depicted values are group means±S.D and corrected by subtraction ofindividual negative controls (untreated BAL cells) which particularlyresulted in negative values. The original IL-4 ELISA results werepositive before correction.

FIG. 4: Technical setup of a VM-device 1 combined with the equineinhalation spacer 2, wherein a 100 ml glass connecting adapter 3 withtwo ground joints, for example, in a 90° angle is connected to themedication holding container 4 attached to the aerosol generator unit 5(nebulizer) e.g. by a wider joint such as a 29/32 joint 6, known to theskilled person. The tighter joint, e.g. a 19/26 joint 7 known to theskilled person, is inserted into a rubber seal 8 on the inlet 9 of theemployed inhalation spacer 2, e.g. a spacer with an inhalation mask 10or an inhalation spacer with nose adapter.

FIG. 5: Release of key cytokines from BALF cells gained fromRAO-affected horses stimulated in vitro by ODN and ODN-GNP before (pre)and after (post) in vivo inhalation therapy, respectively. a, IL-10release from cell cultures stimulated by soluble ODN and ODN-GNP isincreased by inhalation therapy of GNP-bound ODN and subsequent in vitrostimulation (n=18, ±S.D) by 6 different GNP-bound ODNs or by 6 differentsoluble ODNs on average. b, IL-4 release is accordingly decreased.

FIG. 6: Effect of GNP-bound CpG-ODN and plain GNPs on cytokine releasein vivo before and after inhalation therapy. a, IL-10 release isexpressed as n-fold increase based on 1 as initial value beforeinhalation. IL-10 release from RAO-affected horses (n=4, ±S.D.) beforeand after three and five inhalation of GNP-bound CpG-ODN (black bars),from healthy horses (n=4, ±S.D.) before and after three inhalations ofGNP-bound CpG-ODN (grey bars) and from healthy horses (n=4, ±S.D.)before and after three inhalations of plain GNP (white bars). b,IFN-g(amma) release from RAO-affected horses before and after three andfive inhalation of GNP-bound CpG-ODN (black bars) and from healthyhorses before and after three inhalations of plain GNP (white bars).

FIG. 7: Therapeutic effect of GNP-bound CpG-ODNs versus plain GNPs(placebo) on important clinical parameters. a, Breathing rate before andafter treatment by GNP-bound CpG-ODN in RAO-affected horses (n=4,±S.D.), before and after treatment by GNP-bound CpG-ODN in healthyhorses (n=4, ±S.D.) and before and after treatment by GNPs (placebo) inhealthy horses (n=4, ±S.D., left to right). b, Oxygen partial pressurein arterial blood before and after respective treatments. c, Occurrenceof tracheobronchial secrete before and after respective treatments. d,Percentile of neutrophil granulocytes in the TBS before and afterrespective treatments.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of this specification, the term “protein-basednanoparticle”, refers to discrete, dispersible particles in the sizerange 1 to 1000 nm consisting of pharmaceutically acceptable proteins.The proteins and the resulting particles are biocompatible,biodegradable without significant pro-allergenic potential or any toxicproperties, both of the protein and its resulting nanoparticle as wellas of its degradation products.

In the context of this specification, the term “gelatin nanoparticle”(GNP) refers to a protein-based nanoparticle polymerized from gelatinproduced by desolvation. Nanoparticles prepared by a two stepdesolvation technique and subsequent chemical cross-linking (Coester etal. 2000) maintain their non-toxic properties while featuring a higherstorage stability than liposomal liquid formulations and good in vivostability upon administration (Coester et al. 2006).

In the context of this specification, the term “gelatin” refers to anatural polymer derived from porcine, bovine or fish collagen recognizedas a biodegradable and biocompatible starting material being in use asplasma expander for decades (Ward et al. 1977). Alternative, recombinantgelatin can be used. It is obtained mainly by acidic (A-type) oralkaline (B-type), but also thermal or enzymatic degradation of thecollagen. Collagen forms 30% of all vertebrate body protein with amajority in bone and skin. While gelatin and the delivery systems basedon this polymer are biocompatible and biodegradable without toxicdegradation products (Tabata and Ikada 1998; Kawai et al. 2000; Yamamotoet al. 2001; Ward et al. 1977), they are since a long time known forhigh physiological tolerance and low immunogenicity. The overallbeneficial properties of gelatin contributed to its proven record ofsafety as food supplement which is also documented by the classificationas “Generally Recognized as Safe” (GRAS) by the US Food and DrugAdministration (FDA). Moreover, intravenously administered applicationslike plasma expanders (e.g. Gelafundin™, Gelafusal™) consist of gelatinderivatives.

In the context of this specification, the term “lower airways” isdirected to the respiratory brochioles and alveolar sacs (Weibel andGomez 1962) which offer a large surface area for drug interaction andare the stage of several immunologic actions decisive in allergic and/orinflammatory diseases of the respiratory system.

In the context of this specification, the term “CpGoligodeoxynucleotides” refers to short, preferably 20 to 34 bases longartificial, unmethylated sequences featuring cytosine poly guaninepalindromes. They are agonists for a member of the pattern recognitionreceptor (PRR) family signalling through an endosomal membrane basedtype receptor, the Toll-Like Receptor 9 (TLR9) (Angel et al. 2008;Wernette et al. 2002). CpGs were shown to influence several signallingpathways in a variety of immune cells, leading to cytokine production inmany mammalian species (Zwiorek et al. 2004; Zhao et al. 2010). Itappears that the specific purines and pyrimidines surrounding the CpGmotif, phosphothioated backbone, as well as the spacings between CpGmotifs may influence both the level and the type of immune stimulation(Krieg et al. 1995; Mutwiri et al. 2003). CpG motifs improve the antigenpresenting function of dendritic cells (DCs), monocytes and macrophages,induce the proliferation of B lymphocytes, stimulate theimmunoprotective activity of natural killer (NK) cells, and recruit Tcells to the site of ODN administration (Torchilin 2007; Zwiorek et al.2008). Recent studies showed that the immune system responds to CpGmotifs by activating potent Th1-like immune responses which can beharnessed for immune therapy of cancer, allergy, infectious diseases(Krieg 2002), autoimmune diseases, and sterile inflammation (Kanzler etal. 2007). Consequently, CpGs may also be used as potent adjuvant forvaccines in prophylactic anticancer studies involving GNP-bound CpG(Bourquin et al. 2008).

In the context of this specification, the term “A-class” refers to a ODNsequence wherein the CpG are phosphodiesters in palindrome, while the 3′and 5′ tail-guanidines are phosphothioesters, wherein monomers canassociate to tetrads due to G-tail association, and wherein the ODNsequence strongly induce plasmoidal dendritic cell IFN-alpha secretion,while B-cell proliferation is poorly induced. Within the context of thisspecification, the whole sequence backbone may consist ofphosphothioesters.

In the context of this specification, the term “B-class” refers to a ODNsequence wherein the backbone is fully phosphothioate and linear. B-cellproliferation and pDC maturation is strongly induced.

In the context of this specification, the term “C-class” refers to a ODNsequence wherein the backbone is phosphothioate and a 3′ palindromeforms duplexes and wherein the sequence has intermediate effects of bothA- and B-class. In the context of this specification, the term“immunomodulating” refers to the normalization of an otherwise (atopic)disorder of the immune system. By means of the present invention, IL-10levels in the lower airways and in the blood are increased to and/ormaintained at such levels which are typical for individuals notsuffering from any allergic and/or inflammatory disease of the lowerairways. Hence, immunomodulating relates, in consequence, to theamelioration or improvement of clinical symptoms related to allergicand/or inflammatory diseases of the lower airways. Thus,“immunomodulating” has to be distinguished from “immunostimulating”which has to be understood in a way to selectively or unselectivelypromote certain immune cells for action and/or interaction, such asrelease of inflammatory cytokines. In general, an immunomodulator, is asubstance, e.g. a drug, which has an effect on the immune system. Theskilled person is aware of the fact that most drugs do not have effectson only one receptor, so an immunomodulator may be at the same time animmunosuppressant and an immunostimulant, on different targets withinthe immune system. However, in the context of the present application,an immunomodulating composition is understood to comprise tolerogens,such as CpG ODNs which trigger IL-10 release from various immune cells,preferably in the lower airways, and induce regulatory T cells. Theyinduce tolerance and make the tissue, i.e. the lower airways,non-responsive to respective antigens. Tolerance is understood as thestate of non-responsiveness to respective antigens

In the context of this application, a “prolonged” effect relates to ameasurable tolerance which persists even as the treatment has ceased. Indetail, clinical symptoms of an allergic or inflammatory disease of thelower airways remain significantly reduced after treatment has ceased,such as at least 2 weeks after the last medication was administered,preferably 4 weeks, most preferably 12 weeks.

In the context of this specification, the term “nebulizable” refers to acomposition, preferably an aqueous nanoparticle dispersion, whosephysicochemical characteristics are such, that employment of anappropriate nebulizing device, preferably a vibrating mesh nebulizingdevice, leads to a nearly quantitative nebulization efficiency and anoutput rate high enough to guarantee feasible use with an individual.Moreover, the concentration of the nanoparticle dispersion is such thatthe viscosity is not too high to block the nebulizer. Likewise,nebulizable requires that the nanoparticles are small enough not toblock the nebulizer. This includes the absence of agglomerates oraggregates in the nanoparticle dispersion, expressed by a low PDI.

In the context of this specification, the term “vibrating mesh (VM)”refers to passively VM devices such as the Omron Microair nebulizerwhich can be distinguished from active VM devices such as the AeroNeb Goof Nektar (Ghazanfari et al. 2007a). The first group featured aperforated plate with approximately 3 μm-diameter holes. The plate ispassively induced by an attached piezo crystal via a transducer horn.The fluid gets extruded through the microholes and consequently, theaerosol is formed with very high nebulized drug output efficiency and,however, relatively low output rates when viscous formulations wereinvolved. Conversely, the actively VM devices featured a plate withdome-shaped apertures which are moved up- and downwards 10⁵ times persecond in a micrometer range by an electric vibrating element. Thismicropump extrudes the fluid and thus created the aerosol (Ghazanfari etal. 2007a). Described advantages of the active VM device were a morerapid aerosol generation and a relatively high nebulized output over70%.

In the context of the specification, the term “VM nebulization device isattached to the inhalation spacer” refers to a functional combination ofthe VM nebulizer with an inhalation spacer suitable for the individualto receive the composition. To facilitate aerosol delivery in vivo, e.g.a 100 ml glass connecting adapter with two ground joints, for example,in a 90° angle is connected to the medication holding container attachedto the aerosol generator unit (nebulizer) e.g. by a wider joint such asa 29/32 joint, known to the skilled person. The tighter joint, e.g. a19/26 joint known to the skilled person, is inserted into the rubberseal on the inlet of the employed inhalation spacer, e.g. a spacer orinhalation mask for humans or an inhalation spacer with nose adaptersuch as the Euquinehaler® (Equine Health Care, Horsholm, Denmark) or amouth fully covering mask. This combination provided an easy applicabledevice for rapid equine nanoparticulate therapeutics inhalation.

In the context of this specification, the terms “treatment” and“treating” refer to any and all uses which remedy a condition or diseaseor symptoms thereof, prevent the establishment of a condition or diseaseor symptoms thereof, or otherwise prevent or hinder or reverse theprogression of a condition or disease or other undesirable symptoms inany way whatsoever.

In the context of this specification, the term “therapeuticallyeffective amount” includes within its meaning a non-toxic amount ofB-guanidinopropionic acid sufficient to provide the desired therapeuticeffect. The exact amount will vary amongst others from subject tosubject depending on the age of the subject, the gender, the ethnicorigin, their general health, the severity of the disorder being treatedand the mode of administration. It is therefore not possible to specifyan exact “therapeutically effective amount”. However one skilled in theart would be capable of determining a “therapeutically effective amount”by routine trial and experimentation.

In the context of this specification, the term “allergy” is directed totype I hypersensitivities (also called immediate hypersensitivity),which are characterized as rapidly developing reactions of the immunesystem to a trigger. Preferably, said term is directed to essentialhypertension. More preferably, said term is directed to essentialarterial and/or essential pulmonary hypertension.

In the context of this specification, the term “cross-linked” refers tothe stabilization of protein-based nanoparticles produced by desolvationor coacervation. Thereby single protein chains are permanently andcovalently bound together to prevent early disintegration. Cross-linkingmay be facilitated by appropriate enzymes chemical agents.

In the context of this specification, the term “polydispersityindex(PDI)” refers to a dimension-less unit to characterize distributionranges providing normal distribution is given for the data set to beevaluated. Preferably, the PDI is eligible to characterize particle sizedistributions and is used as a quality assessment parameter. A PDI valuebelow 0.1 stands for monodispersity or a very narrow particle sizedistribution, a PDI value between 0.1 and 0.15 stands for a narrowparticle size distribution, a PDI value between 0.15 and 0.25 stands fora broad particle size distribution, while a PDI value between 0.25 and1.0 stands for a very broad particle size distribution orpolydispersity.

In the context of this specification, the term “anti-allergic” isdefined to mean an amount of the nanoparticle-active agent comprisingcomposition that is capable to significantly reduce the clinicalsymptoms and the immunologic parameters of a person with an allergicdisease.

The term “anti-allergic agent” refers to a pharmaceutical compositioncomprising the nanocarrier and the anti-allergic ingredient such as anImmunomodulating CpG ODN which is efficient for use in the treatment ofallergic diseases, especially of those affecting the lower airways andwhich are associated with inflammatory events.

The term “nanocarrier” refers to a pharmaceutically acceptable means inthe nanometer range of formulating the active agent to allow the activeagent to perform its pharmacological action on the desired physiologicalsite.

The term “adjuvant” refers to a pharmaceutically acceptable means ofactively or passively enhancing the active ingredient's interaction withthe desired physiological target.

Preferred Embodiments

Compositions comprising nanoparticles and active agents, preferablynucleic acid active agent, administrated preferably via inhalation, e.g.by a VM device, for use in airway anti-allergic and anti-inflammatorytherapy were not previously reported in prior art.

Previously, jet nebulizers and other devices were used to nebulizeprotein-based nanoparticles, such as GNPs. However, these devicesexercise huge sheer forces on the particle surface and, hence, arepotentially detrimental to sensitive payload such as nucleic acids. Yet,the inventors surprisingly found that nebulizing by VM devices, whichstill exercise some mechanical stress on the nebulized material, istolerated, for example, by sensitive CpG-ODNs of the present inventionwhich retain their immunomodulating properties even after nebulization.Therefore, two established devices with different VM techniques, activeand passive, were compared in addition to the pMDI for GNP integrity,its impact on various parameters' influence of viscosity, its importanceof administration time and recovered concentration after quantitativerecondensation to meet the needs for a convenient application of theinventive composition of the present invention.

Surprisingly, preferably nanoparticulate CpG-ODN compositionsadministrated via inhalation, such as by a nebulization device known inthe art, preferably. by a VM device, most preferably by an active VMdevice, showed an anti-allergic and anti-inflammatory action whenemployed as sole active agent in contrast to prior art, wherein CpG-ODNswere only used as adjuvants, and, even more, not used in inhalationtherapy in a nanoparticulate formulation. Therefore, the inventionrelates to nanoparticulate nucleic acid active agent compositions foruse in the prevention or treatment of allergic and/or inflammatoryairway diseases in an individual, preferably in a mammal, morepreferably in a human, horse, dog, cat, cow, pig, sheep, goat, mouse,rat, gunny pig, elephant, camel, giraffe, hippopotamus and the like,most preferably in a human or horse.

The invention relates to a composition wherein the active ingredient isefficient for use in prevention and/or treatment independent from theallergen. Hence, active agent nucleic acid monotherapy provides theadvantage of antigen-independent treatment of a broad spectrum ofallergic individuals. Thus the composition of the present invention foruse in prevention and/or therapy such as inhalation therapy of allergicand/or inflammatory diseases of the lower airways and the like issuperior over individual antigen desensibilization therapies and thelike in terms of practicability and applicability.

The invention further relates to a method for the prevention(prophylaxis) or treatment of allergic and/or inflammatory airwaydiseases in a subject in need thereof comprising administration to thesubject of a therapeutically effective amount of nanoparticulate nucleicacid active agent compositions, as defined above, wherein the method ischaracterized in that the nanoparticulate nucleic acid active agentcompositions is taken up by competent immune cells and/or epitheliumcells located in the lower airways,

wherein the nanoparticulate nucleic acid active agent compositionscomprise CpG ODNs, preferably class-A, class-B and/or class-C CpG ODNs,more preferably class-A CpG ODNs, even more preferably CpG

wherein the nanoparticulate nucleic acid active agent compositionscomprise the SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and/or14, preferably SEQ ID NO:2. with a fully phosphothioate backbone and/orSEQ ID NO:7 with a full phosphothioate backbone and/or SEQ ID NO:8, 9,10, 11, 12, 13 and/or 14 with a chimerical phosphodiester andphosphothioate backbone (the poly G 3′ and 5′ tail regions), even morepreferably SEQ ID NO:2 with a chimerical phosphodiester andphosphothioate backbone (the poly G 3′ and 5′ tail regions) and/or SEQID NO:8, 9, 10, 11, 12, 13 and/or 14 with a full phosphothioatebackbone.

The pharmaceutical composition of the present invention is eligible foruse in the prevention of aforementioned diseases and associated eventsbecause of its Immunomodulating properties. In the context of theinvention, immunomodulating refers to the normalization of an otherwise(atopic) disorder of the immune system. By means of the presentinvention, IL-10 levels in the lower airways and in the blood areincreased to and/or maintained at such levels which are typical forindividuals not suffering from any allergic and/or inflammatory diseaseof the lower airways.

In a preferred embodiment of the present invention, the protein-basednanoparticles of the pharmaceutical composition are of such a size thatthey are both recognized by the immune competent cells they target inthe lower airways and, at the same time, do neither block the alveolarairways or any other part of the respiratory tract nor cause any otheradverse or repelling reaction. It was shown, that immunomodulatingactivity is best supported on the one hand and nanotoxicological threatsare not an issue on the other hand in the size range of 1 to 1000 nm,preferably 100 to 350 nm, more preferably 150 to 300 nm, most preferably200 to 250 nm, while the PDI is preferably 0 to 0.15 and most preferably0 to 0.1. Hence, the size and size distribution of the nanocarriersdisclosed in the present invention do contribute decisively to theimmunomodulating effect of the surprising composition. The nanocarriersmay also contribute to a sustained release of the active agent at thesite of interaction with competent immune cells in the lower airways.

Pharmaceutical compositions within the scope of the present inventionare preferably for inhalative administration although other ways ofapplication might be possible such as parenteral like intravenous,subcutaneous or intramuscular application of a corresponding sterileformulation or a specialized formulation suitable for transdermal orperoral delivery.

The protein-based nanocarriers of the present invention are preferablynanoparticles made from pharmaceutically acceptable, biocompatible andbiodegradable proteins selected from the group consisting of, albumine,gelatin, legumine, gliadine, elastinlike polypeptide,beta-galactoglobuline and/or silk protein.

Preferably, the protein-based nanocarriers are produced by acoacervation process. Due to relatively mild conditions the coacervationor equally coined desolvation process is the most appropriate andfrequent method to prepare protein-based nanoparticles. Therein, acolloidal system is created when the solvent in which the protein wasinitially dissolved is gradually extracted into an anti-solvent phase.Thereby a phase separation occurs which results in a phase of solidcolloid dispersed in a second phase consisting of the anti-solvent andthe initial solvent (Weber et al. 2000). Consequently, solvent andanti-solvent must be miscible such as water as solvent and ethanol oracetone as anti-solvent whereof the latter was often used to produceGNPs by a two step desolvation technique (Coester et al. 2000; Langer etal. 2003). While a stable size is reached after an initial processperiod the further addition of anti-solvent leads to increased particlesyield in the course of desolvation (Weber et al. 2000). Furthermore, thepH value of the protein solution prior to desolvation has an impact onthe resulting particle size and yield due to higher probability ofprotein coacervation at net-zero surface charge at the isoelectricpoint, which applies to gelatin, HSA, BSA and B-lactoglobulin.

In a preferred embodiment, the composition may comprise active targetingmoieties to target the particles with the active agent to a desired bodysite for specific and/or causal treatment. This may include receptor,antibodies or fragments or hybrids thereof.

In another preferred embodiment, the protein-based nanoparticles arechemically or enzymatically cross-linked. For use in therapeuticapplications, cross-linked nanoparticles are of special importance toguarantee sufficient stability and/or integrity over the required periodof intended activity and/or employment. The protein may be chemicallycross-linked by formaldehyde, dialdehydes, isocyanate, diisocyanate,carbodiimide, alkyldihalogenides and/or glutharaldehyde. Alternatively,enzymatic cross-linking can be performed which is an attractive approachdue to the high specificity of the enzyme catalysis controllable to acertain degree by changing pH and temperature.

Preferably, transglutaminase or laccase is used in the cross-linking ofprotein-based nanoparticles. However, microbial transglutaminase isespecially preferred.

In a very preferred embodiment, glutaraldehyde is used to cross-linkprotein-based nanoparticles (Weber et al. 2000), as no toxic sidereactions or gelatin-associated immunity reactions were observed(Zwiorek 2006). As glutaraldehyde is consumed during the GNPcross-linking reaction and residuals are removed by particlepurification, consequently no adverse effects could be observed.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of the nucleic acid active agent as definedherein into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing into association theβ-guanidinopropionic acid with a nanocarrier and then, if necessary,shaping the product into the desired composition.

Remarkably, the composition of the present invention exhibited aprolonged effect in significantly ameliorating or inhibiting clinicalexpression of allergic and/or inflammatory diseases of the lowerairways. The prolonged effect of at least 4, preferably 12 weeks allowsfor regimen cycles with long regimen-free periods. This administrationscheme increases compliance and is less impairing the patient's qualityof life. In addition, chronic side effects of the composition of thepresent invention are avoided.

Surprisingly, a relatively low dose of the present composition was foundto be effective for use in the prevention and/or treatment of allergicand/or inflammatory diseases of the lower airways. Previous in vivostudies involving CpG as adjuvant ranged from s.c. 100 μg per mouse,i.e. 5000 μg per kg body weight (Bourquin 2008) to 300 μg per human in aclinical trial, i.e. about 4.5 μg per kg body weight (Senti 2009). Inthe present study, significantly lower concentrations were found to beeffective for the above-identified uses, for example 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9 and/or 1.0 μg per kg body weight. The skilledperson appreciates that lower dosage is associated with a lower risk ofside effects, which even allows treatment of such a subpopulation ofpatients which otherwise would not have been eligible for treatmentinvolving an immunomodulating composition. Additionally, lower amountsper dosage result in lower prices per application, which is of advantageespecially with regard to expensive nucleic-acid-based drugs.

Generally, an effective dosage of the nucleic acid active agent presentin the pharmaceutical composition of the present invention is in therange of about 0.0001 to 2 mg per kg body weight, preferably in a rangebetween 0.0001 and 0.01 mg/kg body weight, most preferably in a rangebetween 0.0002 and 0.001 mg/kg body weight. More typically, an effectivedose range is in the range of 0.0001 to 2 mg per kg body weight,preferably in a range between 0.0001 and 0.01 mg/kg body weight, mostpreferably in a range between 0.0002 and 0.001 mg/kg body weight per 72hours; about 0.0005 mg to about 0.05 mg per kg body weight per 72 hours,or about 0.005 mg to about 0.02 mg per kg body weight per 72 hours.

Compositions comprising the nucleic acid active agent or apharmaceutically acceptable salt or derivative thereof may contain anamount of said nucleic acid of 0.1 to 10 wt %, preferably 0.5 to 5 wt %,more preferably 1 to 5 wt % and still more preferably at 5 wt %.

Compositions comprising the nanoparticle-nucleic acid active agentcomposition suitable for oral administration may be present as discretesolid dosage forms such as gelatine or HPMC capsules, cachets orcompressed tablets, each containing a predetermined amount of thenanoparticle-nucleic acid active agent composition, as a powder,granules, as a solution or a suspension in an aqueous liquid or anon-aqueous liquid, or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion.

Compositions for parenteral administration include aqueous andnon-aqueous sterile nanosuspensions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient, and which may includesuspending agents and thickening agents. A parenteral composition maycomprise a cyclic oligosaccaride such as hydroxypropyl-β-cyclodextrin.The compositions may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example saline or water-for-injection,immediately prior to use.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably comprise the nanoparticle-nucleic acid active agent compositionas an optionally buffered aqueous suspension of, for example, 0.01 M to10 M, more preferably 0.05 to 1 M, even more preferably 0.1 M to 0.2 Mconcentration with respect to the compound. Such patches may liberatethe contained pharmaceutical preparation from the reservoir membrane- ormatrix-controlled.

Aerosol compositions for delivery to the lung by inhalation may, forexample be formulated as solutions or suspensions, preferably aqueoussuspensions or solutions and/or suspensions in liquefied propellantdelivered from pressurised packs, such as a metered dose inhaler, withthe use of a suitable liquefied propellant. Suitable propellants includea fluorocarbons or a hydrogen-containing fluorocarbon or mixturesthereof, particularly hydrofluoroalkanes. The aerosol composition may beexcipient free or may optionally contain additional compositionexcipients well known in the art, such as surfactants e.g. oleic acid orlecithin and/or cosolvents e.g. ethanol. Pressurised compositions willgenerally be retained in a canister (e.g. an aluminium canister) closedwith a valve (e.g. a metering valve) and fitted into an actuatorprovided with a mouthpiece.

Alternatively, a dry powder preparation of the nanoparticle-nucleic acidactive agent composition can be used to administer said formulation as adry powder inhaler without propellants and a related suspensionformulation.

Medicaments for administration by inhalation desirably have an optimumparticle size for inhalation into the bronchial system is usually 1-10μm, preferably 1-5 μm to target the alveolar sacs. Particles having asize above 20 μm are generally too large when inhaled to reach the smallairways. When the excipient is lactose it will typically be present asmilled lactose, wherein not more than 85% of lactose particles will havea mass mean diameter (MMD) of 60-90 μm and not less than 15% will have aMMD of less than 15 μm.

Nebulization of the pharmaceutical composition for subsequent inhalationis facilitated preferably by a VM device, more preferably by an activeVM device. Most preferably, this active VM is connected to an inhalationspacer to ease intake of the nebulized pharmaceutical composition,especially for administration to individuals like infants and animals(FIG. 4). Such connection is preferably reversible and is between theactive VM device and is performed by connecting a top preferably 29/32ground joint of a glass connector with two ground joints situated in a90° angle. Moreover, the 19/26 ground joint at the other opening side ofthe glass connector is introduced reversible into the rubber seal of anequine inhalation spacer. The skilled person will acknowledge that jointand/or connector combinations will be required that specifically andexactly fit into the active VM devices employed in every individualcase.

Preferably, the nebulizer in the context of the present invention is anactive VM nebulizer which keeps the nanoparticle-bound delicate nucleicacid active agent in its biologically active form without destroying itby shear forces or other negative influence upon the nebulisationprocess.

The nucleic acid active ingredient is bound to the surface of theprotein-based nanocarrier reversibly via electrostatic interaction.However, interaction is strong enough to keep the nucleic acid activeingredient on the nanocarrier surface until interaction with immunecompetent cells in the lower airways.

The compositions may incorporate a controlled release matrix that iscomposed of sucrose acetate isobutyrate (SAIB) and organic solvent ororganic solvent mixtures. Polymer additives may be added to the vehicleas a release modifier to further increase the viscosity and slow downthe release rate.

The co-administration of the nanoparticle-nucleic acid active agentcomposition or a pharmaceutically acceptable salt thereof or aderivative thereof and one or more further anti-allergic and/oranti-inflammation agents may be simultaneous or sequential. Simultaneousadministration may be effected by nanoparticle-nucleic acid active agentcomposition being in the same unit dose as the anti-allergic and/oranti-inflammation agent, or nucleic acid active agent and theanti-allergic and/or anti-inflammation agent may be present inindividual and discrete unit doses administered at the same, or at asimilar time. Sequential administration may be in any order as required.The anti-allergic and/or anti-inflammation agent may be selected fromthe group consisting of glucocorticoids, H1-antagonists, leukotrieneantagonists, mast cell stabilizers beta2-adrenergic receptor agonists,anti-IgE antibodies or fragments thereof and/or anticholinergic agents,as explained above in detail for the fast acting and long term controlmedication of asthma.

All publications mentioned in this specification are herein incorporatedby reference. The reference in this specification to any priorpublication (or information derived from it), or to any matter which isknown, is not, and should not be taken as an acknowledgment or admissionor any form of suggestion that that prior publication (or informationderived from it) or known matter forms part of the common generalknowledge in the field of endeavour to which this specification relates.

The present invention will now be further described in greater detail byreference to the following specific examples, which should not beconstrued as in any way limiting the scope of the invention.

EXAMPLES Example 1 Preformulation Studies on a NebulizableImmunomodulating Nanoparticulate Composition Materials

Gelatin A Bloom 175, Glutaraldehyde 25% solution, cholamine and EDC werepurchased from Sigma (Taufkirchen, Germany), fluorescent dye Alexa 488was obtained from Invitrogen (Carlsbad, USA). The immunomodulativesingle stranded mixed phosphothioester/-diester backbone CpG-ODN class Awith the sequence 5′-G*G*GGGACGATCGTCG*G*G*G*G*G*-3′ (SEQ ID NO:2) wasreceived as lyophilisate from biomers (Ulm, Germany), diluted insterilely filtrated highly purified water (HPW) produced by a purelabplus device (Elga labwater, Celle, Germany) to a final concentration of1 mg/ml and stored at −80° C. till final use. IL-10 quantifying equineDuo set ELISA was purchased from R&D Systems (Minneapolis, USA).Liquefied gas propellant HFA 134a was obtained from Schick GmbH(Stuttgart, Germany).

Preparation of the CpG-GNPs

Plain, cationized and fluorescent labeled GNPs were prepared accordingto the established protocols (Coester et al. 2000; Zwiorek et al. 2008).20 bases long CpG-ODN 2216 (Biomers, Ulm, Germany) was loaded onto theGNP surface in HPW by electrostatic attraction. To ensure colloidalstability, the CpG concentration was set to 5% (m/m) based on the GNPmass. For this, the aseptically prepared samples of cationized GNPs weresubsequently incubated for 1 h at 22° C. and 300 rpm using aThermomixer™ device (Eppendorf, Hamburg, Germany). The concentration ofthe GNP dispersion was set to 0.5, 1.0 or 1.5 mg/ml for subsequentdifferent nebulization setups.

Determination of Nanoparticle Properties and Dispersion Viscosity

Particle sizes were determined by dynamic light scattering (DLS) using aZetasizer Nano ZS (Malvern Instruments, Worcestershire, England).Nanoparticles were diluted in HPW and measured in concentrations below0.1 mg/ml at 25° C. Thus, any influence of viscosity and of unlikelymultiscattering on the results was ruled out. Intensity weightedparticle mean diameter (Z-average) and polydispersity index (PDI) as thewidth of the fitted Gaussian distribution were calculated by the DTS V.5.10 software from at least 15 subruns. All measurements were performedat least in triplicate. For Zeta potential measurement before and afterloading GNPs were diluted in 10 mM NaCl to maintain a sufficient but nottoo high ionic strength in terms of conductivity and electrodecorrosion. GNP concentration was determined gravimetrically with aMettler Toledo UMX2 (Mettler, Greifensee, Switzerland). Viscosity of GNPformulations was determined by an automated microviscosimeter (AmV)device by Anton Paar GmbH (Graz, Austria).

Percentile loading efficiency was proven indirectly by UV-spectroscopyat 260 nm wavelength (UV1, Thermo Fisher Scientific Inc., Waltham, USA)Therefore, the supernatant(s) of CpG-GNP samples, supernatants of GNPcontrols (without CpG) and supernatants of CpG controls (without GNP)were taken into account as given below:

${{CpG}\mspace{14mu} {loading}} = {\left( {1 - \left( \frac{{{OD}\mspace{14mu} {of}\mspace{14mu} {s\left( {{CpG} - {GNP}} \right)}} - {{OD}\mspace{14mu} {of}\mspace{14mu} {s\left( {{GNP}\mspace{14mu} {control}} \right)}}}{{OD}\mspace{14mu} {of}\mspace{14mu} {s\left( {{CpG}\mspace{14mu} {control}} \right)}} \right)} \right) \times {100\mspace{14mu}\lbrack\%\rbrack}}$

GNP Aerosolization

For aerosolization by pMDIs, 12 ml aluminum pMDI containers were filledwith 3, 5 or 10 g of a 1 mg/ml GNP dispersion and a cap with dosingchamber and purging valves was positioned on top of the container.Crimping and subsequent liquefied propellant filling trough the dosingchamber was conducted by a hand operated laboratory plant 2005/2(Pamasol, Pfaeffikon, Switzerland). The filling weight of propellant waskept constant at 1.5 g/pMDI resulting in 2:1, 4:1 and 8:1 GNPdispersion:propellant ratios. To estimate consistency of dosing, asequence of 30 spraying passes was performed for each formulation andthe pMDI was weighted after each pass. Aerosols and droplets werecollected in suitable 50 ml tubes for subsequent GNP characterization.

VM-nebulization was performed by a passive VM such as NE-U22VMicroair®(Omron, Matsusaka, Japan) and or actively VM such as AeroNebGo® (Aerogen, Galway, Ireland) device. The Passive VM device instrumentwas employed with the manufacturer-provided rubber supplement mouthpiece while the Active VM device was either (a) used with themanufacturer-provided “nebulizer body” connected to the essentialmedication cup/aerosol generator part or with a 90° glass connector withjoints that suitably matched the medication cup/aerosol generator part'soutlet side. Nebulization efficiency (NE) was determined for all threeinstrumental setups by weighing the VM device before nebulization andafter operation to dryness. The latter was considered apparent whenvisibly no more vapor escaped from the aerosol generator. After divisionof the weights, the results were given in percent. To determine thepost-nebulization weight, the whole instrument with all practicallyrelevant adapters or nebulizer bodies was taken into account. Therefore,only those portions of the nebulized formulation that completely escapedthe apparatus and consequently contributed to the deposition study wereconsidered relevant for the NE calculation.

For size and concentration evaluation of post-nebulized GNPs, theresulting aerosol was collected in a closed glass system equipped with awater cooled chiller at 4° C. to quantitatively condensate vapors. Anapplied vacuum of 700 mbars which translated to a flow rate ofapproximately 30 l/min (Vaccubrand CVC200, Wertheim, Germany) wasintroduced to assure high yields of GNP dispersion in the collectionround bottom flask.

Subsequently, intercepted samples were analyzed for size, sizedistribution and concentration. Results were compared to thepre-nebulized ones, respectively. Droplet sizes of dispersions nebulizedby the two VM devices were assessed by laser diffraction. Therefore, 0.5ml of a GNP dispersion (1 mg/ml) were nebulized and the vapor wasdirected through the 633 nm laser beam of a Mastersizer X long bench(Malvern Instruments, Malvern, UK) in 4.5 cm distance to a 300 mm lens.Droplet sizes were calculated by the version 2.19 Mastersizer softwareusing an implemented model based on a particle refractive index of 1.45and a dispersion optical density of 0.276 at 633 nm. Results are themean diameter of three runs each with 1000 measuring events.Corresponding FPFs defined as the particle fraction below 5.21 μm weregiven in percent.

Deposition Study

For deposition studies, fluorescence labeled GNPs with covalently boundAlexa633 dye were employed at a concentration of 1 mg/ml andcharacterized according to Ph. Eur. by apparatus type A twin-stage glassimpinger apparatus (Copley Scientific Ltd., Nottingham, UK,). HPW wasintroduced in the upper (7 ml) and lower (30 ml) stages of the impingerand a steady flow rate of 60 l/min was maintained during theaerosolization process by a Glax. Sing. Sta. pump (Erweka GmbH,Heussenstamm, Germany) to mimic physiologic breathing air flow.Additionally, a 500 ml aerosol spacer (GSK, Brentford, UK) wasintroduced as a pre-separator between the VM-nebulizer or pMDI and theimpinger. FPF to be found in stage 2 of the impinger was considered therespiratory fraction (RF) as part of the whole nebulized fraction.Results of single fractions were calculated by referring the mass perstage calculated from the detected fluorescence intensity of Alexa 633labeled GNPs to the whole applied mass of GNPs in the nebulizer andgiven in percent.

Cell Culture and Immunostimulation

Bronchoalveolar lavage fluid (BALF) was collected from a 20 years oldmale horse of 520 kg body weight with RAO condition sedated bydetomedine (0.01 mg/kg) and butorphanol (0.01 mg/kg) i.v. as previouslydescribed. An endoscope was passed nasotracheally for visual inspectionof the trachea (Hoffman 2008; Jackson et al. 2004). For localanaesthesia, 10 ml of a 2% mepivacaine solution were passed through theendoscope channel and afterwards the endoscope was pulled back uptowards larynx. Subsequently, a BALF catheter (Bivona Inc., Gary, USA)was inserted as far as the bifurcation tracheae, firmly adjusted by aballoon and then employed to introduce two 100 ml aliquots of sterile0.9% NaCl solution. The 200 ml were immediately aspired again by newsterile syringes and transferred to sterile 50 ml centrifugation tubes.Samples were refrigerated at 4° C. shortly until subsequentcentrifugation at 1200 g for 6 minutes to spin down contained cells. Thepellet was resuspended in RPMI medium (Biochrom AG, Berlin, Germany)supplemented with 10% FCS and 67.8 μg/ml penicillin and 113 μg/mlstreptomycin. Cell numbers were counted using a Neubauer chamber(Laboroptik GmbH, Friedrichsdorf, Germany). 2×105 cells per well weretransferred to a 96 well polystyrene cell culture plate (Techno PlasticProducts, Trasadingen, Switzerland) and subsequently incubated witheither nebulized or non-nebulized 0.275 mg GNPs loaded with 13.5 μg (5%(w/w) CpG. Incubation was set to 24 h at 37° C. in a 5% (V/V) CO2atmosphere. Afterwards, culture plates were centrifuged at 1200 g for 6min. and cytokine IL-10 was quantified from collected supernatants by anequine IL-10 ELISA (Duoset, R&D systems, Minneapolis, USA) according tomanufacturer's instructions. Detection wavelength was 450 nm. Remainingcell pellets in the well plate were immediately resuspended in 300 μl of3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)working solution consisting of 82% PBS, 9% FCS and 9% of a 5 mg/ml MTTstock solution. Hence, 0.135 mg MTT reagent was applied per 2×105 cells.After two hours of incubation at 37° C. and in 5% (V/V) CO2 cultureplates were centrifuged at 1200 g for 6 min., supernatants werediscarded and the remaining pellets resuspended in 200 μl DMSO andsubsequently analysed at 590 nm. All samples and related analysis wereconducted in triplicate.

Results

Particle Characterization upon Nebulization by pMDI and VM Devices

First, the impact of nebulization by pMDI was assessed. Therefore, vaporfractions were collected, and PCS measurements revealed initial highparticle sizes and size distributions (PDI values) that diminished inthe course of application. Over a storage time of 48 h, pMDI formulationexhibited no negative impact on unloaded GNPs' stability. As perdifferential weighing, maximum achieved NE with the pMDI was 77.4% forthe 4:1 GNP dispersion:propellant ratio formulation while the others(2:1, 8:1) remained slightly below. After this degree of drawdown,pressure was too low to ensure aerosolization upon liquid's exit fromthe container via the purging valve. Average dosing was 68 mgformulation per pass. However, the S.D of 20 mg indicated a highvariability translating to a relative standard deviation of 29.4%. GNPintegrity was indeed not altered over time by the formulation with HFCpropellant as most likely no interaction occurred between thehydrophilic GNPs and the inert simultaneously hydrophobic and lipophobicliquid gas (Table 1).

TABLE 1 Particle size and size distribution characterized by the PDIvalue of GNPs nebulized by pMDI in dependence of the storage time withinthe pMDI container (n = 3). time point size [h] [nm] S.D. PDI S.D. 0163.8 3.8 0.173 0.036 1.5 150.2 1.3 0.072 0.007 3 152.7 1.8 0.063 0.02848 153.9 2.4 0.076 0.015For unloaded, fluorescing GNPs with an original size of 145.5 nm (±1.76nm) and a very low PDI of 0.038 (±0.025), neither VM-nebulizationprocess altered the assessed parameters significantly (Table 2).

TABLE 2 Sizes and size distributions of plain GNPs after nebulization (n= 3). c [mg/ml] size [nm] S.D. PDI S.D. Passive VM device 0.5 149.91.373 0.027 0.017 1.0 147.4 2.061 0.059 0.035 1.5 146.2 1.601 0.0240.015 Active VM device 0.5 144.9 1.084 0.037 0.025 1.0 144.6 0.989 0.0210.014 1.5 145.6 0.734 0.032 0.023However, CpG-loaded GNPs of another batch being sized 256.2 nm (±3.63nm) pre-nebulization turned out to be significantly smaller at 222.3(±1.42 nm) after nebulization by the passive VM device while not beingsignificantly different at 248.2 (±7.34 nm) with the active VM device.

Nebulization Efficiency of VM Devices

NE remained consistent within the repeated measurements for each singledevice as demonstrated by low S.D. values. Furthermore, the GNPconcentration has hardly any impact on the NE. A negligible tendency ofhigher percentile NE values with rising concentration is visible as itis 93.8, 97.0 and 95.9% and 94.4, 97.0 and 97.8% for the passive VMdevice and for the glass adapter-supplemented active VM device,respectively. However, the applied VM device has an impact. While theabove given values peak near 100%, using the commercial nebulizing body,the active VM device's NE ranks only half at about 50% while the rest istrapped in the device. Recovered masses of re-precipitated samples inthe round flak collector were 70.2, 71.7 and 79.1% (m/m), respectively.

Output Rates of VM-Devices

The passive VM-device required a significantly longer period of time tonebulize constantly employed volume of 1 ml of GNP dispersion completelyto “dryness” compared to the active VM device (Table 3). Viscosity roselinearly with GNP concentration and accordingly output rates dropped.

TABLE 3 Required nebulization times for three GNP concentrations by twoVM devices and related viscosity (n = 3). Active VM Passive VM devicedevice c time time viscosity [mg/ml] [min:sec] S.D. [min:sec] S.D.[mPa * s] S.D. 0.5 01:43 0.001 08:21 0.013 0.9137 0.0003 1.0 01:46 0.00308:49 0.005 0.9201 0.0008 1.5 01:53 0.002 10:57 0.024 0.9283 0.0024

With rising GNP concentration, the output rates were only 0.12, 0.11 and0.09 mg/min, respectively, compared to 4.9 to 5.8 times higher outputrates of 0.58, 0.57 and 0.53 ml/min by the active VM device.

Consistency of Concentration after Nebulization

All three employed GNP concentrations were determined gravimetricallybefore and after nebulization. Post-nebulized concentrations were gainedfrom dispersions precipitated. Results and corresponding deviations ofthe post-nebulized concentration values to the pre-nebulization valuesare given in Table 4. Negative deviations indicating lower GNPconcentrations are found for all three passive VM device runs. For theactive VM device, two deviations were positive indicating aconcentration increase and only one was negative. As a trend, deviationsgot smaller with rising GNP concentration for both VM-devices.

TABLE 4 Comparison of the recovery (absolute and percentile) of threeGNP concentrations before and after nebulization by an active and apassive VM device. Active VM device Passive VM device con- con- con-centration centration deviation centration deviation prior after throughafter through nebu- nebu- nebu- nebu- nebu- lization lization lizationlization lization [mg/ml] [mg/ml] S.D. [%] [mg/ml] S.D. [% ] 0.5 0.5340.057 6.8 0.458 0.003 −8.5 1 0.956 0.131 −4.4 0.974 0.102 −2.6 1.5 1.5160.202 1.1 1.448 0.182 −3.5

Aerosol Particle Size Characterization

Analysis of nebulized droplet sizes revealed slightly higher diametersfor droplets created by the passive VM device. Here, the mean diameterof the active VM device-generated droplets accounted for 6.60±0.03 μmwhile those by the passive VM device was determined at 7.46±0.10 μm(±S.D., n=3). Accordingly, the FPF defined below 5.21 μm (Ghazanfari etal. 2007) was 37.13% (±0.57) and 30.24% (±0.97) for the two VM devices,respectively. When nebulizing HPW alone by the Active VM device, a meandroplet sizes of 6.34 μm and a FPF of 36.28% were received.

Deposition Study

Deposition characteristics were assessed impinger-based to estimate eachpharmaceutical form's feasibility to deliver GNPs in a high RF to thetherapeutically relevant lower airways. GNPs delivered by an pMDIgenerated aerosol showed a bad deposition related to lower airwaytargeting as 65.66% (±0.84%) of the nebulized GNP mass were trapped inthe spacer and only 0.76% (±5.46%) could be found on the last stagerepresenting the RF (FIG. 1). The passive VM device featured aprospective lung deposition of 47.65% (±18.04%) with 1.95% (±0.91%)ending up in the spacer while the active VM-device exhibited the highestRF value of 65.68% (±11.20%) with 3.43 (±0.40%) to be foundpre-separated in the spacer (FIG. 1).

Some particles were deposed on the glass connections and could not bequantitatively assigned to single distinguished stages. They constitutedthe amount lacking to 100%.

Stimulation of IL-10 Release In Vitro

CpG-ODN loading onto the GNPs' surface was 98.51% (±1.29) according tothe gravimetrical differential determination. The Zeta potential wasdetermined to be 23.0 mV (±0.4 mV) before and 21.7 mV (±1.0 mV) afterthe loading. Conductivity dropped accordingly from 0.943 mS/cm (±0.002)to 0.884 mS/cm (±0.003) due to the reduced amount of chargings presentin the dispersion. Once CpGs were successfully loaded onto the GNPs'surface, they were (a) transferred directly without further processingto a cell culture with a defined number of BALF cells or (b) firstnebulized by the active VM-device, completely regained from the vaporand added to the cell culture. After 24 h of incubation, ELISAs wereperformed to determine stimulated cytokine release. Analysis for thecentral immunomodulating cytokine IL-10 revealed significant higherrelease for both the nebulized and the non-nebulized formulationcompared to the control, untreated BALF cells' supernatant. Differencein release between the two applied GNP groups was not significant andaccounted for 225.2 pg/ml (±56.3 pg/ml) and 230.7 pg/ml, respectively(n=3). Additionally, cell viability was very high in both cases,reaching 102.2% (±3.8%) compared to the negative control of amount ofuntreated viable cells as per MTT assay. Plain, non-loaded GNPs did nottrigger IL-10 release in a quantifiable manner and had no negativeeffect on viability over 24 h.

Example 2 Identification of Eligible Nucleic Acid Active Agents andProof of Immunomodulating Principle of the Nanoparticulate CompositionOligodeoxynucleotides

To evaluate the optimal stimulating CpG motif in cultured equine BALcells, three different CpG-ODN classes, with previously employed motifsin horses were compared. Five different CpG-ODNs and one ODN without aCpG motif were selected (Biomers GmbH, Ulm, Germany). Each CpG-ODN classwas represented by two different sequences, except the A-class whereonly one sequence was available. All CpG-ODN classes were singlestranded ODNs with a length of 20 to 30 bases. ODN 2041 (5′-CTG GTC TTTCTG GTT TTT TTC TGG-3′) was used as a CpG-free sequence. The A classdiffers in backbone structure from the other classes. It consists of abackbone chimera of phosphorothioate* (PS) and phosphodiester (PD)modified deoxyribose: CpG-ODN A 2216 (5′-G*G*G GGA CGA TCGTCG*G*G*G*G*G-3′). Two different B-classes were compared: CpG-ODN B 2142(5′-TCG CGT GCG TTT TGT CGT TTT GAC GTT-3′) and CpG-ODN B 2006 (5′-TCGTCG TTT TGT CGT TTT GTC GTT-3′). The C-class was represented by CpG-ODNC 2395: (5′-TCG TCG TTT TCG GCG CGC GCC G-3′) and CpG-ODN C M362:(5′-TCG TCG TCG TTC GAA CGA CGT TGA T-3′). A working concentration of2.5 mg/ml for GNPs and 1.0 mg/ml for ODNs was used. In brief, 76.5 μl ofGNP stock solution was diluted by 230 μl highly purified water (HPW) andmixed with 43.8 μl of CpG-ODN stock solution by gentle stirring toobtain a final ODN concentration of 0.125 mg/ml. As references, 0.125mg/ml CpG-ODN solution of each class (306.5 ml HPW, 43.8 μl CpG-ODNstock solution) and 2.5 mg/ml GNP dispersion (274 μl HPW, 76.5 μl GNPstock solution) were prepared. All the samples were incubated for 90minutes at room temperature with constant stirring at 300 rpm in aThermomixer (Eppendorf, Hamburg, Germany). Thereafter, samples wereready for use. The samples were stored at 4° C. and used within 48hours. Five different CpG-ODN sequences of three different classes andone ODN lacking a CpG motif, as described above, were incubated with theequine BAL cells in triplicate. In detail, 0.275 mg GNPs loaded with13.5 μg (5% (w/w)) ODN or 13.5 μg of soluble ODN were added per well tocompare the effects of unbound ODNs and ODNs bound to GNPs. To estimatethe immunostimulating response of ODNs in cell culture, supernatant wastaken after 24 hours of incubation and analyzed by equine ELISAs (R&DSystems, Minneapolis, USA). Three key-cytokines namely IL-4, IL-10 andIFN-γ were evaluated. The ELISAs were performed according to themanufacturer's protocol. Limits of detection of the applied ELISA assayswere 15.6-2000, 156.25-20000 and 31.2-4000 pg/ml, respectively. Givenvalues were corrected by subtraction of untreated BAL cells, whichserved as a negative control.

Cell Viability by MTT-Assay

MTT assays were performed to evaluate cell viability followingincubation with CpG-ODN and GNP-bound CpG-ODN. After removal ofsupernatant cell pellets were immediately resuspended in 300 μl MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) workingsolution consisting of 82% PBS, 9% FCS and 9% of a 5 mg/ml MTT stocksolution. Then 0.135 mg MTT reagent was added per 2×10⁵ cells. After twohours of incubation at 37° C. and in 5% (V/V) CO2 the culture plateswere centrifuged at 1200 g for 6 min. The supernatant was discarded andthe remaining pellets were resuspended in 200 μl DMSO to solubilizeviolet formazan crystals. The absorbance of each well was measured at530 nm using wallac Victor² 1420 multilabel counter (Perkin Elmer,Fremont, Calif., USA). Untreated BAL cells served as a reference for100% viability. All measured experiments were conducted in triplicate.

Quantification of BAL Cell Subpopulations

To evaluate percentile fraction of BAL cell subpopulations (macrophages,lymphocytes and neutrophile granulocytes) from RAO-affected and healthyhorses 1 ml of every BAL cell-pellet was centrifuged two times with 1200g for 6 min on glass sides. Theses cytospots were stained withdiff-quick staining set (Medion diagnostics, Düdingen, Switzerland) and300 cells respectively were counted manual under microscope. Thepercentile fraction of macrophages, lymphocytes and neutrophilegranulocytes was identified; mean values out of three countsrespectively were identified.

Cytokine Release in Cell Culture Upon Stimulation by CpG-ODN/GNP-BoundCpG-ODN IL-10 Release (Only FIG. 2)

In order to compare the effect of GNP-bound CpG-ODN versus solubleCpG-ODN on cytokine release from BAL cells, six sequences of ODNs (toinclude five CpG-ODNs) were tested. Throughout, IL-10 showed the highestrelease in absolute concentration values of the three quantifiedcytokines. As a general observation, cells from healthy horses secretedsignificantly (P=0.0047) higher amounts of IL-10 than those ofRAO-affected horses. In FIG. 2 a IL-10 stimulation of soluble CpG-ODN isshown. Out of the six different ODNs, CpG 2216 (A-class) showed thehighest IL-10 release for both examined groups. IL-10 release was 400pg/ml in cells from healthy horses and 220 pg/ml in those from RAOhorses. The B and C class triggered only low levels of IL-10 release inRAO-derived cells, with concentrations of about 125 pg/ml. C-classesappeared to result in higher stimulation (350 pg/ml) than B-class (200pg/ml) in cell culture from healthy horses. The employed B-classes (ODN2142 and ODN 2006) and C-classes (ODN M362 and ODN 2395) showed almostidentically release behavior in cell culture from RAO horses. In FIG. 2b IL-10 stimulation by GNP-bound CpG-ODNs is shown. Most noticeable wasa significant higher release of IL-10 of the cells from healthy horses(P=0.0051) compared to RAO-affected horses. Furthermore the GNP-boundCpG-ODN 2216 (A-class) induced the highest IL-10 concentration (540pg/ml) which surpassed the release value by soluble CpG-ODNs as shown inFIG. 2 a. On the contrary the stimulation by other particle-boundCpG-ODNs (B- and C-class) was lower than those by soluble CpG-ODN.

IL-4 Release (only FIG. 3)

In contrast to IL-10, IL-4 release upon stimulation by six soluble ODNsdid not differ (P=0.614) between RAO and healthy horses derived cells(FIG. 3 a). Both absolute values were low and inter and intra variationswere high. However, as shown in FIG. 3 b, difference of highsignificance (P=0.001) was found when comparing GNP-bound CpG-ODNstimulated cell cultured from RAO and healthy horses for IL-4 release.The latter resulted in negative values (FIG. 3 b) while those ofRAO-affected horses were positive and not distinguishable from thoseprovoked by soluble CpG-ODN (P=0.9469). Furthermore GNP-bound CpG-ODNlowered IL-4 release of cells of healthy horses considerably more thansoluble CpG-ODN (P=0.0018) (FIG. 3 a).

IFN-γ Release

IFN-γ release from RAO and healthy horses derived cell cultures isdisplayed after stimulation by soluble CpG-ODNs. On average, nosignificant difference could be observed between the mean values ofcells from RAO-affected and healthy horses (P=0.3514). However, the sixemployed ODNs revealed distinctive effects. As seen for IL-10, thehighest release was induced by CpG 2216 A-class and accounted for 94±10pg/ml in RAO and 135±19 pg/ml in healthy horses derived cell cultures.All other ODNs induced lower amounts between 15 and 50 pg/ml. A tendencytowards higher release by healthy horse derived cells could be assumedbut was not statistically significant. In contrast, GNP-bound ODNs ledto a clear discrimination between RAO and healthy derived cells in termsof IFN-γ (P=0.008). Here, CpG 2216 A-class stimulated the highest IFN-γsecretion as well, accounting for 76±14 pg/ml (healthy) and 25±10 pg/ml(RAO). In this individual example (CpG 2216), a marginally statisticallynot significant difference was found (P=0.05). Also, no or slightrelease from RAO cell cultures was found, while no statisticalsignificance occurred between the healthy derived cell cultures treatedby CpG-ODN in comparison with GNP-bound CpG-ODN (P=0.45).

MTT Assay

BAL cells from RAO-affected and healthy horses were used to investigatein vitro cell viability after 24 h of incubation with regard todetectable differences after administration of soluble CpG-ODNs comparedto GNP-bound CpG-ODNs. Four mean values (cells from RAO horses on theone hand treated with CpG-ODN or with GNP-bound CpG-ODN and cells fromhealthy horses on the other hand treated with CpG-ODN or GNP-boundCpG-ODN), were averaged out of the individual viability results of thesix employed ODNs. Untreated BAL cells were assessed as 100% viabilityreference. In the healthy group no significant differences weredetectable in viability between soluble CpG-ODN and GNP-bound CpG-ODNadministration. No significant differences were seen between the sixODNs examined individually. Of all evaluated means of the four groups,lowest viability was observed in cells from RAO-affected horses treatedwith soluble CpG-ODN (69.7%±6.6%). In comparison, cells fromRAO-affected horses with GNP-bound CpG-ODN showed the highest viability,on average 104.3%±6.4%, which was significantly higher (P<0.0001). As anexample, difference between cells treated with CpG-ODN 2216 A-class andcells treated with GNP-bound CpG-ODN 2216 A-class was found to besignificant (P=0.0405). However, mean viability of cells from healthyhorses exposed to soluble CpG-ODN was 94.2%±4.0% and with GNP-boundCpG-ODN administration 91.7%±8.0%. No significant differences wereobserved between these two mean values (P=0.512). Accordingly, nosignificant difference was identified in the CpG-ODN 2216 A-classexample (P=0.5319). Cells derived from RAO-affected and healthy horsesdid not differ in viability when challenged GNP bound CpG-ODN 2216A-class (P=0.808). Summarizing, BAL cells gained from RAO-affectedhorses showed significant higher cell viability in MTT assay whenincubated with GNP-bound CpG-ODN in comparison to soluble CpG-ODN. Thisdifference was not seen in assays with cells gained from healthy horses.

With regard to the results, the optimal stimulating sequence among fiveexplored CpG-ODNs in equine BAL cells was the A-class 2216. Thedetectable IL-10 upregulation was higher than expected as CpG-ODNs werepreviously known for potent IFN-γ release in general and CpG-ODN A-classfor IFN-α release in particular (Krieg, 2002). With regard to theobservation that no significant difference of IL-10 release within B-and C-class by cells from RAO-affected horse was detectable, we couldhypothesize that the class is more determining than the individualCpG-ODN sequence.

Example 3 Preliminary Clinical Study as Proof of Principle in aTherapeutic Clinical Setting Nebulization of the ImmunomodulatingNanoparticulate Composition

For inhalation studies, an Equine Haler™ spacer (Equine HealthCare Aps,Hoersholm, Denmark) and an active VM device were combined by a 90° glassconnector with ground joints that suitably matched the aerosol generatorpart's outlet diameter and the spacer's inlet. An identical protocol wasrun for negative control placebo trial and for the medication trial. Thenegative control exclusively contained an aqueous (HPW) GNP dispersion(1.5 mg/ml) while the medication was a combination of GNP (1.5 mg/ml)and CpG-ODN 2216 (0.075 mg/ml). Healthy and RAO-affected horses wereinhaled three times alike with two-day intervals between individualadministrations followed by a control BAL. Two additional subsequentinhalations and one final BALF examination for disease developmentmonitoring were added if significant changes occurred after the thirdinhalation. Clinical examinations, blood gas analysis, endoscopicexploration and cytology of TBS were performed at the beginning, afterthree and finally after five inhalations.

Clinical Examination and Lung Scoring

A lung scoring system was further developed comprising clinicalparameters (nasal discharge, breathing rate), blood gas chemistry,endoscopic exploration, cytology of tracheo-bronchial secret (TBS) andof bronchoalveolar lavage fluid (BALF). Accordingly, 12 horses of a meanweight of 477.7 kg and aged 12.0 years on average were scored. The studywas approved by the regional legal agency for animal experiments(Regierung von Oberbayern, Munich, Germany) and designated the approvalcode 55.2-1-54-2531-31-10. The applied scoring system allowed groupingthe patients into four categories (healthy, mild, moderate and severeRAO). For the clinical trial, three groups of horses were established,with the first group (n=4) consisting of healthy horses (mean age of 8.8years) for the placebo negative control, the second group (n=4)consisting of healthy horses for compatibility study (mean age of 10.4years) and the third group (n=4) of moderate RAO-affected horses fortherapeutic efficiency verification (mean age of 16.8 years). The keyarterial blood gas parameter of partial pressure of oxygen (PaO₂) wasmeasured by a Radiometer Copenhagen NPT 7 series (Radiometer GmbH,Willich-Schiefbahn, Germany). Physiological values for PaO₂ were set to100 mmHg (±5 mmHg). Moreover, percentages of neutrophil granulocytes outof total cell count from TBS cytology were calculated after staining byDiff-Quick® staining set (Medion diagnostics, Diidingen, Switzerland).Physiological range of breathing rate was defined as 8 to 16 breaths perminute while higher values were considered as pathological.

Results (Only FIGS. 5 to 7)

To evaluate the efficiency of envisaged inhalation therapy, BALF wasobtained before and after the regimen from RAO-affected and healthyhorses. Data from healthy individuals served as physiological reference.BALF cells were stimulated in vitro by six different ODNs. In FIG. 5 ashows in vitro IL-10 expression of cells derived from RAO-affectedhorses treated by ODNs and GNP-bound ODNs both before and afterinhalation. After inhalation treatment, a significantly (P<0.0001)higher IL-10 release (390 pg/ml) in BAL cell cultures stimulated byGNP-bound ODNs was observed as compared to the state before inhalationtreatment (83 pg/ml) (FIG. 5 a). Similar trend of IL-10 release wasobserved after stimulation (389 pg/ml) by soluble ODNs (FIG. 5 a)compared to the value of 139 pg/ml before inhalation regime (P=0.0002).Accordingly, IL-4 in vitro expression was decreased significantly afterinhalations of both soluble (P=0.0298) and GNP-bound (P=0.0282) ODNs(FIG. 5 b).

Overall, IFN-γ release in vitro was low and did not reveal a generaltrend after treatment by GNP-bound ODNs (P=0.1414) or by soluble ODNs(P=0.4870) before (pre) versus after (post) inhalation treatment ofRAO-affected horses. FIG. 6 a clearly depicts the increase of IL-10expression detected in BALF supernatant in RAO-affected horses. Whilethree inhalations led to a significant 3.8-fold increase (P=0.0473) inIL-10 expression, a 6.9-fold increase was found after five inhalations(FIG. 6 a). Therefore, the average IL-10 levels differ significantly(P=0.034) before starting and after finishing the full five inhalationregimen applied to RAO-affected horses. Healthy horses exhibited a 2.14fold augmentation in IL-10 expression after pulmonary administration ofGNP-bound CpG-ODN confirming the principle of action (FIG. 6 a).However, differences in expression levels before and after inhalationwere marginally statistically significant (P=0.089). In contrast, nosignificant difference (P=0.289) was found when comparing healthy horsesbefore and after three pulmonary administrations of blank GNPs which wasgiven as placebos (FIG. 6 a). In vivo secretion of IL-4 and IFN-γ wasanalyzed in BALF supernatants before and after inhalation regimens. IL-4levels were below detection threshold in vivo. For IFN-γ, a significantimpact of GNP-bound CpG-ODN regimen was observed. FIG. 6 b reveals aconstant increase after three and five consecutive inhalations comparedto IFN-γ levels in BAL supernatants before the regimen (P=0.0034).Placebo administration did not result in altered cytokine expression(P=0.8322) (FIG. 6 b) while IFN-γ data could not be obtained fromhealthy individuals treated with GNP-bound CpG-ODN.

Firstly, the breathing rate per minute was assessed to discriminatehealthy and RAO-affected individuals. The latter exhibited a breathingrate of 19.6 (±1.47) breaths per minute (bpm) before treatment (FIG. 7a) which was significantly higher than the measured value 13.6 (±0.98)bpm of healthy horses (P=0.0094). The regimen (five doses) lowered therate significantly down to 12.8 (±0.80) bpm (P=0.0036) (FIG. 7 a).

Healthy horses had a PaO₂ of 94 mmHg (±2.07) (FIG. 7 b). In contrast,RAO-affected horses showed a PaO₂ of 86.75 mmHg (±2.29) (FIG. 7 b). Thismean value was significantly improved (P=0.0153) towards 95.6 mmHg(±1.69) by the full regimen of five inhalations (FIG. 7 b). Thereafter,no statistically significant difference was observed compared with thehealthy animals (P=0.5384). The percentage of neutrophil granulocyteswithin the TBS of RAO-affected horses was high (70%±0.50) beforetreatment and differed significantly from values of healthy horsesexhibiting 26% (±6.0) (P=0.0004) (FIG. 7 d). Treatment by GNP-boundCpG-ODN contributed to a significant decrease down to 50% (±2.04) bythree inhalations (P<0.0001) and down to 40% (±6.52) by five inhalations(P=0.0048), respectively (FIG. 7 d). After fife applications ofGNP-bound CpG-ODN no statistically significant difference could beobserved compared to healthy horses (P=0.195). Furthermore, FIG. 7 dshows that in healthy horses neither the GNP-bound CpG-ODN (P=0.3472)nor the placebo (P=0.8171) resulted in a significant change ofneutrophil percentages, respectively.

Beside cytokine-based immunologic parameters, the clinical impact of thehereby proposed therapy was assessed. No local or systemic adverseeffects after inhalation of GNP-bound CpG-ODN (CpG-GNP) were observedindicating the good biocompatibility of applied doses and regime. Thisis the first time an in vivo application of nanoparticle-boundimmunostimulating DNA via inhalation in horses is reported in thisstudy.

The inhalation regimen lowered the breathing rate of RAO-affected horsessignificantly and can therefore be regarded efficient.

The determination of partial pressure of blood oxygen (PaO₂) was used toevaluate the extent of gas exchange and the response to treatment. Themagnitude of gas exchange abnormality correlates with the severity ofbronchiolitis and clinical signs.

Within TBS, the percentage of neutrophil granulocytes represents astrong indicator for RAO. Moreover, it is regarded as one of the mostdecisive parameters to evaluate RAO. As a consequence an averagedecrease of 40% of neutrophils in TBS cytology after fife inhalationswith GNP-bound CpG-ODN could be estimated as one of the most importantclinical ameliorations after this immunotherapy. The percentage ofneutrophils within the TBS was directly related to the severity of theRAO condition. Therefore, it can be deducted that the severity of thepathogenesis was significantly reduced after five applications ofGNP-bound CpG-ODN

Previously, T_(reg) activation was related to a reduction in activityand the number of neutrophile granulocytes by promoting their rate ofapoptosis. Human neutrophils express all known TLRs except TLR3.Moreover, TLRs were shown to possess a crucial impact on T_(reg)stimulation and function. Previous investigations confirmed that T_(reg)cells inhibit neutrophils other than by direct cell-cell contactmechanism (CTLA-4/B7-1 mechanism) and especially through IL-10 action.Furthermore, this mechanism was advantageous in the treatment ofallergic diseases. Therefore, it is concluded that the observedimpressive IL-10 induction by the hereby proposed treatment can bedirectly related to the decreasing neutrophils' percentage and thereforedirectly contributes to the regimen's anti-RAO effectiveness.Consequently, further studies with higher patient numbers and doseescalation are in preparation in order to elucidate the full potentialof this first applied inhalative nanoparticle based immunotherapy.

Example 4 Evaluating the Long Term Effect of the Present Composition onSymptoms of Allergic and/or Inflammatory Diseases of the Lower AirwaysMethod

24 horses suffering from an allergic and inflammatory disease affectingthe lower airways, i.e. RAO, enrolled in a placebo-controlled clinicalstudy evaluating the long-term efficiency of the immunomodulatingnanoparticulate composition of the present invention. The individualswere examined for decisive parameters associated with the allergic andinflammatory disease affecting the lower airways prior the treatmentcycle with comprised 5 inhalations each separated by a treatment-freeday. Clinical examination was further performed 1d and 28 d after thelast administration. The immunomodulating nanoparticulate compositionwas administered by VM-facilitated nebulization via a combined spacer asdepicted in FIG. 4.

Results and Discussion

Decisive clinical parameters such as partial oxygen blood pressure andbreathing frequency neared physiologic values, i.e. they weresignificantly increased and decreased in contrast to placebo treatment,respectively. Further clinical parameters such as trachea mucus quantityand viscosity, coughing, nasal discharge, bifocatio and percentage ofneutrophils in the tracheal mucus could be significantly lowered,leading to tremendous improvement for the quality of life of theaffected individuals. Importantly, the observed improvements were foundsignificant even after 28 d from the last administration indicating along-lasting therapeutic effect.

1. A pharmaceutical composition comprising a pharmaceutically acceptablepolymerized protein-based nanocarrier and a preventative or therapeuticamount of an active agent, for use in the prevention and/or treatment ofan allergic and/or inflammatory disease of the lower airways in amammal, wherein the active agent is an oligonucleotide and/or anoligodeoxynucleotide (ODN) which is effective for use in the preventionand/or treatment of an allergic and/or inflammatory disease of the lowerairways, wherein the ODN is selected from the group consisting ofguanidine phosphodiester cytosine (CpG) ODN class A, class B and/orclass C, and wherein the active agent is coupled to the polymerizedprotein-based nanoparticle in a manner wherein the active agentmaintains its preventive and/or therapeutic activity.
 2. Thepharmaceutical composition according to claim 1, wherein the polymerizedprotein-based nanocarrier is a gelatin nanoparticle, an albuminnanoparticle, a legumine nanoparticle, a gliadine nanoparticle, anelastinlike polypeptide nanoparticle, a beta-galactoglobulinenanoparticle and/or a silk protein nanoparticle.
 3. The pharmaceuticalcomposition according to claim 1, wherein the pharmaceutical compositionis a nebulizable aqueous dispersion or a nebulizable aqueous dispersionmade from a lyophilisate.
 4. The pharmaceutical composition according toclaim 1, wherein the aqueous dispersion is nebulized by a vibrating meshnebulization device, wherein the resulting droplet size is preferably inthe size range between 1 to 5 μm, wherein the respirable dropletfraction is 50 to 100%, wherein the nebulization efficiency is more than95%, wherein the nanoparticle concentration within the aqueousdispersion is preferably in the range between 1.0 to 2.0 mg/ml, whereinthe dispersant in the aqueous dispersion is highly purified water with aconductivity below 0.55 μS/cm or a physiological 0.9% NaCl solution,wherein the aqueous dispersion has a viscosity in the range between 0.85and 1.1 mPa/s. and wherein the nebulizable aqueous dispersion isnebulized by a vibrating mesh nebulization device at an output rate over0.5 ml/min.
 5. The pharmaceutical composition according to claim 1,wherein the comprised oligonucleotide and/or ODN is administered at alow, side-effects-avoiding dose of 0.0001 to 2 mg per kg body weight,preferably in a range between 0.0001 and 0.01 mg/kg body weight, mostpreferably in a range between 0.0002 and 0.001 mg/kg body weight.
 6. Thepharmaceutical composition according to claim 1, wherein the CpG ODNcomprises a central palindrome CG motif-containing sequence flanked by apolyG sequence and a chimerical phosphodiester/phosphorothioatebackbone.
 7. The pharmaceutical composition according to claim 1,wherein the CpG ODN is selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3 SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, and/or SEQ ID NO:14 and/or a derivative thereof comprising achimerical phosphodiester/phosphorothioate backbone or a fullphosphorothioate backbone.
 8. The pharmaceutical composition accordingto claim 1, wherein the CpG induces the release of immunomodulatingcytokine interleukin-10.
 9. The pharmaceutical composition according toclaim 1, wherein the mammal is a human, a primate, or a domestic animalor an animal for production, preferably a horse.
 10. The pharmaceuticalcomposition according to claim 1, wherein the amount of oligonucleotideand/or of ODN is 0.1 to 10 wt %, preferably 1 to 7.5 wt % and morepreferably 5 wt % in relation to the nanoparticle mass.
 11. Thepharmaceutical composition according to claim 1, wherein thepharmaceutical composition further comprises excipients selected fromthe group consisting of buffer salts, surfactants, lyoprotectors, dyes,chelating agents and/or radionuclides.
 12. The pharmaceuticalcomposition according to claim 1, wherein the pharmaceutical compositionis administrated to an individual via inhalation, wherein a vibratingmesh nebulization device is combined with an inhalation spacer in such amanner that the individual can inhale the nebulized pharmaceuticalcomposition from the inhalation spacer, and wherein the vibrating meshnebulization device is attached to the inhalation spacer, so that thenebulized pharmaceutical composition is quantitatively available to theindividual at the inhalation spacer's top part nasal outlet.
 13. Thecomposition of claim 1, wherein the pharmaceutical composition induces aprolonged clinical effect by reducing or inhibiting allergic and/orinflammatory symptoms and/or events in the lower airways and/or in thegeneral state of health of the treated individual.
 14. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is administered 1 time per day, 2 times per day, 3 times perday, 4 times per day, 5 times per day, 6 times per day, 1 time per week,2 times per week, 3 times per week, 4 times per week, 5 times per weekor 6 times per week, 3 times per months, 2 times per months or 1 timeper month.
 15. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is administered in a cycle comprising aperiod of 3 to 14 d with 3 to 7 applications of the composition of claim1, followed by an application-free period of 7 to 84 d.
 16. Thepharmaceutical composition according to claim 1 comprising a furtherantiantiallergic and/or anti-inflammatory active agent selected from thegroup consisting of glucocorticoids, H1-antagonists, leukotrieneantagonists, mast cell stabilizers beta2-adrenergic receptor agonists,and/or anticholinergic agents.
 17. Method for the production of thepharmaceutical composition according to claim 1 comprising the steps ofcontacting the nanocarrier with the active ingredient in a ratio of 90to 99% nanocarrier to 1 to 9% active agent in a pharmaceuticallyacceptable medium, sensitive mixing, incubating for 0.5 to 6 h,purification and adjusting the concentration to a nebulizablenanosuspension.